The mechanism of DNA-operator recognition by repressor and cro proteins of bacteriophage lambda, 434 and P22 will be analyzed by a combination of crystallographic structural studies and directed mutagenesis. The structure of a cocrystal of the amino-terminal domain of 434 repressor with its complete 14 base pair operator will be determined to about 3 A (the limit of diffraction) using standard crystallographic methods. Directed mutagenesis will be used to generate modified 434 and lambda repressors and cros in order to test and extend models for recognition based on the crystal structure. The binding specificities and regulatory properties of the altered proteins will be examined. Efforts to crystallize some of the modified proteins, as well as to crystallize 434 repressor alone and 434 cro with and without DNA will be made, since these structures are essential to a complete picture of operator binding. Modified operators will likewise be constructed. Mechanisms of positive control in lambda will be studied by directing changes in residues implicated in repressor-polymerase contact by pc mutations, and structures of potentially informative modified repressors will be studied by difference Fourier analysis when possible. This group of proteins presents an outstanding opportunity for understanding regulatory interactions of the proteins with each other and with DNA. We anticipate that, ultimately, it will be possible to create repressors with entirely new specificities.